Mesenchymal stem cell and the method of use thereof

ABSTRACT

Demyelinated axons were remyelinated in the demyelinated rat model by collecting bone marrow cells from mouse bone marrow and transplanting the mononuclear cell fraction separated from these bone marrow cells.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Divisional of U.S. patent application Ser. No.13/040,954, filed Mar. 4, 2011, which is a Continuation of applicationSer. No. 12/076,092, filed Mar. 13, 2008, which is a Continuation ofapplication Ser. No. 11/189,050, filed Jul. 26, 2005, now abandoned,which is a Divisional of application Ser. No. 10/330,963, filed Dec. 23,2002, now U.S. Pat. No. 7,098,027, which is a Continuation-In-Part ofPCT/JP01/05456, filed Jun. 26, 2001, the contents of all of which areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to cells derived from bone marrow cells,cord blood cells, or embryonic hepatic tissues that can differentiateinto neural cells, and cell fractions containing such cells. It isexpected that these cells and cell fractions can be used to treatneurological diseases, particularly in autologous transplantationtherapy.

BACKGROUND ART

Transplantation of oligodendrocytes (i.e., oligodendroglia) (Archer D.R., et al., 1994. Exp. Neurol. 125:268-77; Blakemore W. F., Crang A. J.,1988. Dev. Neurosci. 10:1-11; Gumpel M., et al. 1987. Ann. New YorkAcad. Sci. 495:71-85) or myelin-forming cells, such as Schwann cells(Blakemore W. F., 1977. Nature 266:68-9; Blakemore W. F., Crang A. J.,1988. Dev. Neurosci. 10:1-11; Honmou O. et al., 1996. J. Neurosci.16:3199-208), or olfactory ensheating cells (Franklin R. J. et al.,1996. Glia 17:217-24; Imaizumi T. et al., 1998. J. Neurosci.18(16):6176-6185; Kato T. et al., 2000. Glia 30:209-218), can elicitremyelination in animal models and recovery of electrophysiologicalfunction (Utzschneider D. A. et al., 1994. Proc. Natl. Acad. Sci. USA.91:53-7; Honmou O. et al., 1996. J. Neurosci. 16:3199-208). It ispossible to prepare such cells from patients or other persons for celltherapy. However, this method is considerably problematic because tissuematerial must be collected from either the brain or nerves.

Neural progenitor cells or stem cells derived from brain have theability to self-renewal and differentiate into various lineages ofneurons and glia cells (Gage F. H. et al., 1995. Proc. Natl. Acad. Sci.USA. 92:11879 83; Lois C., Alvarez-Buylla A., 1993. Proc. Natl. Acad.Sci. USA. 90:2074-7; Morshead C. M. et al., 1994. Neuron 13:1071-82;Reynolds B. A., Weiss S., 1992. Science 255:1707-10).

By transplantation into newborn mouse brain, human neural stem cellscollected from fetal tissues differentiate into neurons and astrocytes(Chalmers-Redman R. M. et al., 1997. Neurosci. 76:1121-8; Moyer M. P. etal., 1997. Transplant. Proc. 29:2040-1; Svendsen C. N. et al., 1997.Exp. Neurol. 148:135-46), and myelinate the axons (Flax J. D. et al.,1998. Nat. Biotechnol. 16:1033-9). Remyelination and recovery of impulseconduction upon transplantation of neural progenitor (stem) cellsderived from adult human brain into demyelinated rodent spinal cord havebeen reported (Akiyama Y. et al., 2001. Exp. Neural.).

These studies have evoked great interest due to the indicatedpossibility of the application of the above-mentioned cells toregenerative strategy of neurological diseases (Akiyama Y. et al., 2001.Exp. Neural.; Chalmers-Redman R. M. et al., 1997. Neurosci. 76:1121-8;Moyer M. P. et al., 1997. Transplant. Proc. 29:2040-1; Svendsen C. N. etal., 1997. Exp. Neurol. 148:135-46; Yandava B. D. et al., 1999. Proc.Natl. Acad. Sci. USA. 96:7029-34). However, in order to establish celltransplantation therapy (including autologous transplantation) usingthese cells, still problems, such as establishment of harvest method andrequirement of cell expansion using trophic factors, remain to besolved.

According to the recent studies, neural stem cells were revealed to beable to differentiate or transform into hematopoietic cells in vivo,suggesting that neural progenitor (stem)-cells are not restricted to theneural cell lineage (Bjornson C. R. et al., 1999. Science 283:534-7).Furthermore, bone marrow stromal cells (not mesenchymal stem cells inthe bone marrow) are reported to differentiate into astrocytes by theinjection into the lateral ventricles of neonatal mice (Kopen G. C. etal., Proc. Natl. Acad. Sci. USA. 96:10711-6), and into neurons in vitrowhen cultured under appropriate cell culture conditions (Woodbury D. etal., 2000. J. Neurosci. Res. 61:364-70).

DISCLOSURE OF THE INVENTION

The present inventors have previously isolated and cultured neural stemcells from adult human brain, and established some cell lines. Bystudying their functions, the inventors newly discovered that the neuralstem cells have pluripotency and the ability to self-renewal.Specifically, single-cell expansion of neural progenitor (stem) cellsobtained from adult human brain was conducted to establish cell lines;the established cells were then subjected to in vitro clonal analysis.The result showed that the cell lines had pluripotency (namely,differentiation into neuron, astroglia (or astrocyte), andoligodendroglia (i.e., oligodendrocyte)) and the ability to self-renewal(namely, proliferation potency). Thus, these cells were confirmed topossess the characteristics of neural stem cell.

Transplantation of these cells indeed resulted in very favorable graftsurvival, migration, and differentiation in cerebral ischemic model ratsor injury model rats. Furthermore, transplantation of the cells wasfound to result in functional myelin sheath formation in spinal corddemyelination model rats. Thus, such transplantation allowsremyelination of the demyelinated axon and restoration of the neuralfunction in the rat spinal cord demyelination model. Effectiveness ofsuch transplantation therapy using these cells was confirmed byhistological, electrophysiological, and behavior studies.

Judging from the above-described findings, transplantation of culturedneural stem cells, which have been isolated from a small amount ofneural tissue collected from the cerebrum of a patient, into the lesionof the brain or the spinal cord of the patient seems to be a widelyapplicable in autotransplantation therapy.

However, while not causing neurologic deficits, collecting tissuescontaining neural stern cells from cerebrum is relatively invasive.Thus, considering the need for establishing therapeutic methods forvarious complicated diseases in the nervous system today, it is crucialto establish a safer and simpler method for autotransplantation therapy.

Thus, an objective of the present invention is to provide cellularmaterial that is useful in the treatment of neurological diseases, andwhich can be prepared safely and readily. Another objective of thepresent invention is to provide a method for treating neurologicaldiseases, preferably a method for autotransplantation therapy, using thecellular material.

In view of the existing state as described above, to establish donorcells the present inventors focused on the technique of collecting bonemarrow cells from bone marrow, a simpler technique as compared to thecollection of neural stem cells and routinely used in today's medicalpractice. First, they collected bone marrow cells from mouse bonemarrow, isolated mononuclear cell fraction, and then transplanted thisfraction as donor cells into spinal cord demyelination model rats.Surprisingly, it was discovered that the demyelinated axon getsremyelination by the treatment. Hence, the present inventors newlyrevealed that the mononuclear cell fraction prepared from bone marrowcells have the ability to differentiate into neural cells. The presentinventors also discovered that cell fractions containing mesodermal stemcells, mesenchymal stem cell, stromal cells, and AC133-positive cells,that were isolated from the mononuclear cell fraction had the ability todifferentiate into neural cells. Besides bone marrow cells, these cellfractions can also be prepared from cord blood cells. Furthermore,AC133-positive cells can be prepared from embryonic hepatic tissues.

Thus, the present invention provides cell fractions containing cellscapable of differentiating into neural cells, which are isolated frombone marrow cells, cord blood cells, and embryonic hepatic tissues.

In another embodiment, such cell fractions contain mesenchymal stemcells having the following character: SH2(+), SH3(+), SH4(+), CD29(+),CD44(+), CD14(−), CD34(−), and CD45(−).

In another embodiment, such cell fractions contain stromal cells havingthe following characteristics: Lin(−), Sca-1(+), CD10(+), CD11D(+),CD44(+), CD45(+), CD71(+), CD90(+), CD105(+), CDW123(+), CD127(+),CD164(+), fibronectin (+), ALPH(+), and collagenase-1(+).

In another embodiment, such cell fractions contain cells having thecharacter AC133(+).

In addition, the present invention provides cells capable ofdifferentiating into neural cells, which are contained in theabove-mentioned cell fraction.

Furthermore, the present invention provides compositions for treatingneurological disease, which contain the above-mentioned mononuclear cellfractions or the above-mentioned cells. According to a preferredembodiment of the present invention, the neurological disease isselected from the group consisting of: central and peripheraldemyelinating diseases; central and peripheral degenerative diseases;cerebral apoplexy (cerebral infarction, cerebral hemorrhage,subarachnoid hemorrhage); brain tumor; dysfunction of higher function ofthe brain (the term “higher function of the brain” involves thecognitive function, short and long memory, speech, etc.); psychiatricdiseases; dementia; infectious diseases; epilepsy; traumaticneurological diseases; and infarction of spinal cord diseases.

Furthermore, the present invention provides therapeutic methods forneurological diseases, which comprises transplanting of theabove-mentioned mononuclear cell fractions, cells, or compositions. Inpreferred embodiments, the donor cells are derived from a recipient.

The present invention provides mononuclear cell fractions isolated frombone marrow cells, cord blood cells, or embryonic hepatic tissues,wherein the fractions contain cells capable of differentiating intoneural cells. It is unclear whether the differentiation of cellscontained in the cell fractions provided by the present invention intoneural cells is caused by the transformation of so-called hematopoieticcells into neural cells, or, alternatively, by the differentiation ofimmature cells capable of differentiating into neural cells that arecomprised in bone marrow cells, etc. However, the majority of the cellsdifferentiating into neural cells are assumed to be stem or precursorcells, namely, cells having the self-renewal ability and pluripotency.Alternatively, the cells differentiating into neural cells may be stemor precursor cells which have differentiated to some extent intoendoderm or mesoderm.

Cells in a cell fraction of the present invention do not have to beproliferated with any trophic factors (then again they can proliferatein the presence of trophic factors). Thus, these cells are simple andpractical from the standpoint of the development of autotransplantationtechnique for the diseases in the neural, and are very beneficial inmedical industry. In general, a cell fraction of the present inventionis derived from vertebrate, preferably from mammal (for example, mouse,rat, human, etc.).

A cell fraction of the present invention can be prepared by subjectingbone marrow cells or cord blood cells collected from vertebrate todensity-gradient centrifugation at 2,000 rpm in a solution for asufficient time ensuring separation depending on specific gravity, andthen recovering the cell fraction with a certain specific gravity withinthe range of 1.07 to 1.1 g/ml. Herein, the phrase “a sufficient timeensuring separation depending on specific gravity” refers to a timesufficient for the cells to shift to a position in the solutionaccording to their specific gravity, which is typically about 10 to 30minutes. The specific gravity of the cell fraction to be recovered iswithin the range of 1.07 to 1.08 g/ml (for example, 1.077 g/ml).Solutions, such as Ficoll solution and Percoll solution, can be used forthe density-gradient centrifugation, but is not limited thereto.

Specifically, first, bone marrow (5 to 10 μl) collected from avertebrate is combined with a solution (2 ml L-15 plus 3 ml Ficoll), andthen centrifuged at 2,000 rpm for 15 minutes to isolate a mononuclearcell fraction (approx. 1 ml). The mononuclear cell fraction is combinedwith culture solution (2 ml NPBM) to wash the cells, and then the cellsare again centrifuged at 2,000 rpm for 15 minutes. Then, theprecipitated cells are recovered after the removal of the supernatant.Besides femur, sources to obtain a cell fraction of the presentinvention include sternum, and ilium constituting the pelvis. Any otherbone can serve as a source so long as it is large enough. A cellfraction of the present invention can also be prepared from bone marrowsand cord blood stored in bone marrow bank or cord blood bank.

Another embodiment of cell fractions of the present invention includes amononuclear cell fraction isolated and purified from bone marrow cellsor cord blood cells, which contains mesodermal (mesenchymal) stem cellscapable of differentiating into neural cells. The term “mesodermal(mesenchymal) stem cell” refers to cells that can copy (divide andproliferate) cells with the same potential as themselves and that arecapable of differentiating into any type of cells constitutingmesodermal (mesenchymal) tissues. Mesodermal (mesenchymal) cellsindicate cells constituting tissues that are embryologically categorizedto the mesoderms, including blood cells. The mesodermal (mesenchymal)stem cell includes, for example, cells characterized by SH2(+), SH3(+),SH4(+), CD29(+), CD44(+), CD14(−), CD34(−), and CD45(−). A cell fractioncontaining mesodermal (mesenchymal) stem cells can be obtained, forexample, by selecting cells having a cell surface marker, such as SH2,as described above from the above-mentioned cell fraction obtained bycentrifuging bone marrow cells or cord blood cells (the cell fractionaccording to claim 2).

Furthermore, a cell fraction containing mesodermal (mesenchymal) stemcells capable of differentiating into neural cells can be prepared bysubjecting bone marrow cells or cord blood cells collected fromvertebrate to density-gradient centrifugation at 900 G in a solution fora sufficient time ensuring separation depending on specific gravity, andthen recovering the cell fraction with a certain specific gravity withinthe range of 1.07 to 1.1 g/ml. Herein, “a sufficient time ensuringseparation depending on specific gravity” refers to a time sufficientfor the cells to shift to a specific position corresponding to theirspecific gravity in the solution for density-gradient centrifugation,which is typically about 10 to 30 minutes. The specific gravity of acell fraction to be recovered varies depending on the type of animal(for example, human, rat, and mouse) from which the cells have beenderived. A solution to be used for density-gradient centrifugationincludes Ficoll solution and Percoll solution, but is not limitedthereto.

Specifically, first, bone marrow (25 ml) or cord blood collected fromvertebrate is combined with an equal volume of PBS solution, and thencentrifuge at 900 G for 10 minutes. Precipitated cells are mixed withPBS and then are recovered (cell density=approx. 4×10⁷ cells/ml) toremove blood components. Then, a 5-ml aliquot thereof is combined withPercoll solution (1.073 g/ml), and centrifuged at 900 G for 30 minutesto extract a mononuclear cell fraction. The extracted mononuclear cellfraction is combined with a culture solution (DMEM, 10% FBS, 1%antibiotic-antimycotic solution) to wash the cells, and is centrifugedat 2,000 rpm for 15 minutes. Finally, the supernatant is removed,precipitated cells are recovered and cultured at 37° C. under 5% CO²atmosphere.

Another embodiment of a cell fraction of the present invention is afraction of mononuclear cells isolated from bone marrow cells or cordblood cells, which contains stromal cells capable of differentiatinginto neural cells. Examples of stromal cell include cells characterizedby Lin(−), Sca-1(+), CD10(+), CD11D(+), CD44(+), CD45(+), CD71(+),CD90(+), CD105(+), CDW123(+), CD127(+), CD164(+), fibronectin (+),ALPH(+), and collagenase-1(+). A cell fraction containing stromal cellscan be prepared, for example, by selecting cells having a cell surfacemarker, such as Lin as described above, from the above-mentioned cellfraction obtained by centrifuging bone marrow cells or cord blood cells(the cell fraction according to claim 2).

Furthermore, such a cell fraction can be prepared by subjecting bonemarrow cells or cord blood cells collected from vertebrate todensity-gradient centrifugation at 800 G in a solution for a sufficienttime ensuring separation depending on specific gravity, and thenrecovering the cell fraction with a certain specific gravity within therange of 1.07 to 1.1 g/ml. Herein, “a sufficient time ensuringseparation depending on the specific gravity” indicates a timesufficient for the cells to shift to a specific position correspondingto their specific gravity in the solution for density-gradientcentrifugation, which is typically about 10 to 30 minutes. The specificgravity of a cell fraction to be recovered is preferably within therange of 1.07 to 1.08 g/ml (for example, 1.077 g/ml). A solution to beused for density-gradient centrifugation includes Ficoll solution andPercoll solution, but is not limited thereto.

Specifically, first, bone marrow or cord blood collected from vertebrateis combined with an equal volume of a solution (PBS, 2% BSA, 0.6% sodiumcitrate, and 1% penicillin-streptomycin). A 5-ml aliquot thereof iscombined with Ficoll+Paque solution (1.077 g/ml) and centrifuged at 800G for 20 minutes to obtain a mononuclear cell fraction. The mononuclearcell fraction is combined with a culture solution (Alfa MEM, 12.5% FBS,12.5% horse serum, 0.2% i-inositol, 20 mM folic acid, 0.1 mM2-mercaptoethanol, 2 mM L-glutamine, 1 μM hydrocortisone, 1%antibiotic-antimycotic solution) to wash the cells, and then arecentrifuged at 2,000 rpm for 15 minutes. The supernatant is removedafter centrifugation. The precipitated cells are collected and thencultured at 37° C. under 5% CO² atmosphere.

Another embodiment of a cell fraction of the present invention is amononuclear cell fraction containing cells characterized by AC133(+)capable of differentiating into neural cells, which is isolated frombone marrow cells, cord blood cells, or embryonic hepatic tissues. Sucha cell fraction can be obtained, for example, by selecting cells havinga cell surface marker including the above-mentioned AC133(+) from thecell fraction obtained, as described above, by centrifuging bone marrowcells or cord blood cells (the cell fraction according to claim 2).

Furthermore, the cell fraction can be obtained by subjecting embryonichepatic tissues collected from vertebrate to density-gradientcentrifugation at 2,000 rpm in a solution for a sufficient time ensuringseparation depending on specific gravity, recovering a cell fractionwithin the range of a specific gravity of 1.07 to 1.1 g/ml, and thenrecovering cells with the characteristic of AC133(+) from the cellfraction. Herein, “a sufficient time ensuring separation depending onspecific gravity” indicates a time sufficient for the cells to shift toa specific position corresponding to their specific gravity in thesolution for density-gradient centrifugation, which is typically about10 to 30 minutes. The solution to be used for density-gradientcentrifugation includes Ficoll solution and Percoll solution, but is notlimited thereto.

Specifically, first, liver tissue collected from vertebrate is washed inL-15 solution, and then treated enzymatically in an L-15 solutioncontaining 0.01% DNaseI, 0.25% trypsin, and 0.1% collagenase at 37° C.for 30 minutes. Then, the tissue is dispersed into single cells bypipetting. The single-dispersed embryonic hepatic cells are centrifugedby the same procedure as described for the preparation of themononuclear cell fraction from femur in Example 1(1). The cells thusobtained are washed, and then AC133(+) cells are collected from thewashed cells using an AC133 antibody. Thus, cells capable ofdifferentiating into neural cells can be prepared from embryonic hepatictissues. The antibody-based recovery of AC133(+) cells can be achievedusing magnetic beads or a cell sorter (FACS, etc.).

Transplantation of any of these cell fractions containing mesodermalstem cells, mesenchymal stem cells, stromal cells, or AC133-positivecells into demyelinated spinal cord can lead to efficient remyelinationof the demyelinated region. In particular, the above-mentioned cellfraction containing mesenchymal stem cells can engraft favorably anddifferentiate into neural cells such as neurons or glia whentransplanted into a stroke model or a cerebral infarction model.

The present invention also provides cells capable of differentiatinginto neural cells, which are contained in the above-mentioned cellfraction. These cells include, for example, neural stem cells,mesodermal stem cells, mesenchymal stem cells, stromal cells, andAC133-positive cells which are contained in the above-mentioned cellfraction, but are not limited thereto so long as they can differentiateinto neural cells.

The present invention also provides compositions for treatingneurological diseases, which comprise a cell fraction or cells of thepresent invention. It is possible to transplant the cell fractions orcells of the present invention without modification. However, in orderto improve the efficiency of therapy, they may be transplanted ascompositions combined with various additives or introduced with genes.The preparation of compositions of the present invention may comprise,for example, (1) addition of a substance that improves the proliferationrate of cells included in a cell fraction of the present invention orenhances the differentiation of the cells into neural cells, orintroducing a gene having the same effect; (2) addition of a substancethat improves the viability of cells in a cell fraction of the presentinvention in damaged neural tissues, or introducing a gene having thesame effect; (3) addition of a substance that inhibits adverse effectsof damaged neural tissue on the cells in a cell fraction of the presentinvention, or introducing a gene having the same effect; (4) addition ofa substance that prolongs the lifetime of donor cells, or introducing agene having the same effect; (5) addition of a substance that modulatesthe cell cycle, or introducing a gene having the same effect; (6)addition of a substance to suppress the immunoreaction or inflammation,or introducing a gene having the same effect; (7) addition of asubstance that enhances the energy metabolism, or introducing a genehaving the same effect; (8) addition of a substance that improves themigration of donor cells in host tissues, or introducing a gene havingthe same effect; (9) addition of a substance that improves blood flow(including inductions of angiogenesis), or introducing a gene having thesame effect; (10) addition of a substance that cure the infectiousdiseases, or introducing a gene having the same effect, or (11) additionof a substance that cure the tumors, or introducing a gene having thesame effect, but is not limited thereto.

It is considered that the cells according to the present invention areimmobilized in the bone marrow by a distinct mechanism involving acertain type of substance (proteins, etc.) and do not normally move outinto the peripheral blood. Therefore, to make these cells enter theperipheral blood circulation, conventionally, they are removed from thebone marrow, and then administered intravenously. However, the studiesconducted by the present inventors gradually elucidated the mechanism ofimmobilization of these cells in the bone marrow. The discovery made bythe present inventors showed that these cells, which had been localizedin the bone marrow, could be made to move out into the peripheral bloodby intravenous injection of active factors, such as an antibody, acytokine, chemicals, or a growth factor. That is, therapeutic effect ofbone marrow transplantation that is similar to that of theaforementioned method can be expected from intravenous injection of anactive factor, such as an antibody, a cytokine, chemicals, or a growthfactor.

A cell fraction, cell, and composition of the present invention can beused for treating neurological diseases. Target neurological diseasesfor the therapy include, for example, central and peripheraldemyelinating diseases; central and peripheral degenerative diseases;cerebral apoplexy (including cerebral infarction, cerebral hemorrhage,and subarachnoid hemorrhage); cerebral tumor; disorders of higher brainfunction including dementia; psychiatric diseases; epilepsy, traumaticneurological diseases (including head injury, cerebral contusion, andspinal cord injury); infectious diseases; and infarction of spinal cord,but are not limited thereto.

According to the present invention, cells derived from bone marrow cellsof a recipient can be transplanted as donor cells (autotransplantationtherapy). This has the following advantages: (1) low risk of rejectionfor the transplantation; and (2) no difficulty in usingimmunosuppressant. When autotransplantation therapy is arduous, thencells derived from other person or nonhuman animal may be used. Cellsfrozen for storage are also usable. The donor cells may be derived fromcord blood.

Bone marrow can be collected, for example, by anesthetizing (by local orsystemic anesthesia) an animal (including human) that serves as asource, put a needle into the sternum or iliac of the animal, andaspirating the bone marrow with a syringe. On the other hand, it is anestablished technique to collect cord blood at birth by putting needledirectly into the umbilical cord, and aspirating the blood from theumbilical cord using syringe, and to store the blood.

Transplantation of cells into a patient can be performed, for example,by first filling a syringe with cells to be transplanted. Herein, thecells are suspended in an artificial cerebrospinal fluid orphysiological saline. Then, the damaged neural tissue is exposed bysurgery, and, with a needle, directly injecting the cells into thelesion. Due to high migrating potential of cells contained in a cellfraction of the present invention, they can migrate in the neuraltissues. Hence, cells can be transplanted into a region adjacent to thelesion. Moreover, injection of the cells into the cerebrospinal fluid isalso expected to be efficacious. In the case of the injection of thecells into the cerebrospinal fluid, the cells can be injected into apatient by typical lumbar puncture, without surgical operation onlyunder local anesthetization. Thus, the patient can be treated inpatient's sickroom (not in an operation room), which makes the methodpreferable. Alternatively, intravenous injection (including any systemictransplantations such as intravenous, intraarterial, selectiveintraarterial administration) of the cells is also expected to beeffective. Thus, transplantation can be carried out by a procedure basedon typical blood transfusion, which is advantageous in that thetreatment can be performed in patient's sickroom.

Furthermore, due to their high migrating potential, cells in a cellfraction of the present invention can be used as a carrier (vector) forgenes. For example, the cells are expected to be useful as a vector forgene therapy for various neurological diseases such as brain tumor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows optical light micrographs of transections of dorsal columnof the spinal cord, which had been prepared at 1-mm intervals. (A),normal axon; and (C), damaged demyelinated axon. Patterns of dorsalcolumn observed at higher magnification are shown in (B) for normal axonand in (D) for demyelinated axon. Scale bars: 250 μm in (A) and (C); 10μm in (B) and (D).

FIG. 2 shows microphotographs demonstrating the remyelination of ratspinal cord (A), after transplantation of adult mouse bone marrow cells;and (C), after transplantation of Schwann cells. Photomicrographsdemonstrating remyelinated axon observed at higher magnification areshown in (B), after transplantation of bone marrow cells; and (D), aftertransplantation of Schwann cells. Scale bars: 250 μm in (A) and (C); 10μm in (B) and (D).

FIG. 3 shows an electron micrograph of remyelinated axon after thetransplantation of bone marrow into the dorsal columns. The tissue wastreated with substrate X-Gal to detect transplanted bone marrow cellscontaining the β-galactosidase gene in the damaged tissues (the reactionproduct is indicated with arrow). When observed at higher magnification,basal lamina was found around the fibers (wedge-shaped mark; scale bar 1μm). The presence of large cytoplasmic and nuclear regions and basallamina in the transplanted cells indicates peripheral myelination.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described in more detail below withreference to examples based on specific experiments.

Example 1 Preparation of Bone Marrow Cells and Schwann Cells

(1) Bone Marrow Mononuclear Cells

Mouse bone marrow cells (10 μl) were obtained from the femur of adultLacZ (a structural gene of β-galactosidase) transgenic mice (The JacksonLaboratory, Maine, USA). The collected sample was diluted in L-15 medium(2 ml) containing 3 ml Ficoll, and centrifuged at 2,000 rpm for 15minutes. Cells were collected from the mononuclear cell fraction, andsuspended in 2 ml serum-free medium (NPMM: Neural Progenitor cellMaintenance Medium). Following centrifugation (2,000 rpm, 15 min), thesupernatant was removed, and precipitated cells were collected andre-suspended in NPMM.

(2) Schwann Cells

Primary Schwann cell cultures were established from the sciatic nerve ofneonatal mouse (P1-3) according to the method of Honmou et al. (J.Neurosci., 16(10): 3199-3208, 1996). Specifically, cells were releasedfrom sciatic nerve by enzymatic and mechanical treatment. 8×10⁵ cellsper plate were plated onto 100-mm² poly (L-lysine)-coated tissue cultureplates and the cells were cultured in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% (vol/vol) fetal calf serum.

Example 2 Experimental Animal Preparation and Transplantation

(1) Preparation of Demyelinated Rat Model

Experiments were performed on 12 week old Wistar rats. A localizeddemyelinated lesion was created in the dorsal columns using X-rayirradiation and ethidium bromide injection (EB-X treatment).Specifically, rats were anesthetized with ketamine (75 mg/kg) andxylazine (10 mg/kg) i.p., and a surface dose of 40 Grays of X-ray wasirradiated using Softex M-150 WZ radiotherapy machine (100 kV, 1.15 mA,SSD 20 cm, dose rate 200 cGy/min) on the spinal cord caudal to the tenththoracic spine level (T-10) through a 2×4 cm opening in a lead shield (4mm thick). Three days after X-ray irradiation, rats were anesthetized asabove, and aseptic laminectomy of the eleventh thoracic spine (T-11) wasconducted. A demyelinating lesion was generated by the direct injectionof ethidium bromide (EB) into the dorsal columns via a glassmicropipette whose end was drawn. 0.5 saline containing 0.3 mg/ml EB wasinjected at the depths of 0.7 and 0.4 mm.

(2) Transplantation of Stem or Progenitor Cells that can Differentiateor Transform into the Neural Lineages

3 days after the EB injection, 1 μl of the cell suspension (1×10⁴cells/μl), which was obtained in Example 1, was injected into the middleof the EB-X-induced lesion. Transplanted rats were immunosuppressed withcyclosporin A (10 mg/kg/day).

Example 3 Histological Examination

Rats were deeply anesthetized by the administration of sodiumpentobarbital (60 mg/kg, i.p.), and perfused through the heart cannula,first, with phosphate-buffer solution (PBS) and then with a fixativecontaining 2% glutaraldehyde and 2% paraformaldehyde in 0.14 MSorensen's phosphate buffer, pH 7.4. Following in situ fixation for 10minutes, the spinal cord was carefully excised, cut into 1 mm segmentsand kept in fresh fixative. The tissue was washed several times inSorensen's phosphate buffer, post-fixed in 1% OsO₄ for 2 hours at 25°C., dehydrated by elevating the concentration of the ethanol solution,passed through propylene oxide and embedded in EPON. Then, the tissuewas cut into sections (1 μm), counterstained with 0.5% methylene blueand 0.5% azure II in 0.5% borax, and examined under light microscope(Zeiss: Axioskop FS). Ultrathin sections were counterstained with uranyland lead salts, and examined with JEOL JEM1200EX electron microscope(JEOL, Ltd., Japan) at 60 kV.

A 50×50 μm standardized region in the central core of the dorsal columnsin the spinal cords near the site wherein the cells were initiallyinjected was used for morphometric analysis. The numbers of remyelinatedaxons and cell bodies associated with the axons were counted within thisregion; and the density to square millimeters was calculated.Furthermore, the diameters of the axons and cell bodies, the number ofcells with multi-lobular nuclei, and cells showing myelination wereexamined in the same standardized region. Measurements and counts wereobtained from five sections per rat, and five rats (n=5) were analyzedfor each experimental condition. All variances represent standard error(±SEM).

The dorsal column in the spinal cord mostly consists of myelinated axons(FIG. 1A, B). The proliferation of endogenous glial cells was inhibitedby the irradiation of X-ray to the dorsal columns of the lumbar spinalcord. Further, by the administration of a nucleic acid chelator,ethidium bromide, glial cells such as oligodendrocytes were founddamaged and local demyelination occurred. Such lesions generatedaccording to this procedure are characterized by almost complete loss ofendogenous glial cells (astrocytes and oligodendrocytes) andpreservation of axons (FIG. 1C). Examination of the lesion with a lightmicroscope under a higher magnification revealed that congested areasconsisting of demyelinated axons are appositioned closely to one anotherseparated by areas wherein the debris of myelin exist and whereinmacrophages exist (FIG. 1D). The lesion occupied nearly the entiredorso-ventral extent of the dorsal columns 5 to 7 mm longitudinally.Almost none of the endogenous invasion of Schwann cells or astrocytesoccurs till the sixth to eighth week. With the start of invasion, theseglial cells begin to invade into the lesion from peripheral borders.Thus, a demyelinated and glial-free environment is present for at least6 weeks in vivo.

Three weeks after transplantation of LacZ transgenic mouse bone marrowcells (BM) into the central region of the lesion in immunosuppressed anddemyelinated rat models, extensive remyelination of the demyelinatedaxons was observed (FIGS. 2A and 2B). Remyelination was observed acrossthe entire coronal dimension of the dorsal columns and throughout theantero-posterior extent of the lesion. FIGS. 2C and 2D show the patternof remyelination observed after transplantation of allogeneic Schwanncells (SC) into the EB-X lesion model. It is remarkable that largenuclear and cytoplasmic regions were found around the remyelinatedaxons, a characteristic of peripheral myelin, by both BM and SCtransplantations.

Example 4 Detection of β-Galactosidase Reaction Products In Vivo

Three weeks after transplantation, β-galactosidase expressingmyelin-forming cells were detected in vivo. Spinal cords were collectedand fixed in 0.5% glutaraldehyde in phosphate-buffer for 1 h. Sections(100 μm) were cut with a vibratome and β-galactosidase expressingmyelin-forming cells were detected by incubating the sections at 37° C.overnight with X-Gal (substrate which reacts with (β-galactosidase todevelop color) at a final concentration of 1 mg/ml in X-Gal developer(35 mM K₃Fe (CN)₆/35 mM K₄Fe (CN)₆3H₂O/2 mM MgCl₂ in phosphate-bufferedsaline) to form blue color within the cell. Sections were then fixed foran additional 3 h in 3.6% (vol/vol) glutaraldehyde in phosphate-buffer(0.14 M), and were examined with light microscope for the presence ofblue reaction product (β-galactosidase reaction product). Prior toembedding in EPON, the tissue was treated with 1% OsO₄, dehydrated in agraded series of ethanol, and soaked in propylene oxide for a shortperiod. Ultrathin sections were then examined under an electronmicroscope without further treatment.

Under the electron microscope, most of the myelin-forming cells derivedfrom donor cells retained the basal membrane (FIG. 3: wedge-shapedmark). Additionally, the myelin-forming cells had relatively largenucleus and cytoplasm, which suggests the formation of a peripheralnervous system-type myelin sheath.

It was confirmed that almost no endogenous remyelination byoligodendrocytes or Schwann cells occurs for at least six weeks in thelesion model used in the present experiment. Furthermore, the donorcells that contained the reporter gene LacZ, i.e., X-Gal-positive cells,were observed to form myelin at the electron microscopic level (FIG. 3:arrow).

Differentiation into neurons and glial cells could be observed followingthe transplantation of bone marrow cells into the EB-X lesions, but notby SC transplantation. Five percent of lacZ-positive cells (transplantedbone marrow cells) in the EB-X lesions showed NSE (NeuronSpecific-Enolase)-immunoreactivity and 3.9% showed GFAP (Glial FibriallyAcidic Protein)-immunoreactivity, indicating that some of the bonemarrow cells can differentiate into neurons or glial cells,respectively, in vivo.

Furthermore, employing antibodies, the present inventors isolatedmesenchymal stem cells with the characteristic of cell markers SH2(+),SH3(+), CD29(+), CD44(+), CD14(−), CD34(−), and CD45 (−) from the cellfraction obtained in Example 1 (1). Furthermore, they discovered thattransplantation of the cells into the demyelinated regions of rat spinalcord results in more efficient remyelination. It was also revealed thatthe cells survived favorably and differentiated into neurons or neuronalcells and glia cells when transplanted into cerebral infarction modelrats.

Further, the present inventors isolated stromal cells characterized bythe cell surface markers Lin (−), Sca-1(+), CD10(+), CD11D(+), CD44(+),CD45(+), CD71(+), CD90(+), CD105(+), CDW123(+), CD127(+), CD164(+),fibronectin (+), ALPH(+), and collagenase-1(+) from the cell fractionobtained in Example 1 (1). Transplantation of the cells intodemyelinated regions of rat spinal cord also resulted in efficientremyelination.

Further, the present inventors isolated cells characterized by the cellsurface marker AC133 (+) from the cell fraction obtained in Example 1(1). Transplantation of the cells into demyelinated regions of ratspinal cord also resulted in efficient remyelination.

In addition, the present inventors obtained a cell fraction containingAC133-positive cells capable of differentiating into neural cells fromrat embryonic hepatic tissues by the following procedure. Specifically,first, liver tissues collected from rat fetuses were washed in L-15solution, and then treated enzymatically in L-15 solution containing0.01% DNaseI, 0.25% trypsin, and 0.1% collagenase at 37° C. for 30minutes. Then, the tissue was dispersed into single cells by pipettingseveral times. The single-dispersed embryonic hepatic tissues werecentrifuged as in Example 1 (1) (preparation of mononuclear cellfraction from femur) to isolate a mononuclear cell fraction. Theobtained mononuclear cell fraction was washed, and then, AC133(+) cellswere recovered from the cell fraction using anti-AC133 antibody. Theisolation of AC133-positive cells can be achieved using magnetic beadsor a cell sorter (FACS or the like). Transplantation of the obtainedAC133-positive cells into demyelinated regions of rat spinal cord alsoresulted in efficient remyelination.

INDUSTRIAL APPLICABILITY

As described above, the present invention provides fractions ofmononuclear cells isolated and purified by collecting bonemarrow-derived bone marrow cells, cord blood-derived cells, or fetalliver-derived cells. Transplantation of such mononuclear cell fractionsinto a demyelination model animal was confirmed to result inremyelination of the demyelinated axon.

Cells for transplantation can be'relatively easily isolated from a smallquantity of bone marrow cell fluid aspirated from bone marrow, and canbe prepared for transplantation in several tens of minutes after thecells are being collected. Thus, these cells can serve as useful andregenerable cellular material for autotransplantation in the treatmentof demyelinating diseases.

This invention highlights the development of the autotransplantationtechnique to treat demyelinating diseases in the central nervous system.Furthermore, the use of the present invention in transplantation andregeneration therapy for more general and diffuse damage in the nervoussystem is envisaged. In other words, this invention sheds light onautotransplantation therapy against ischemic cerebral damage, traumaticcerebral injury, cerebral degenerating diseases, and metabolicneurological diseases in the central and peripheral nervous systems.

According to the present invention, cells in the hematopoietic system(bone marrow or cord blood) are used as donor cells. Thus, to treatneurological diseases, the cells may be transplanted into the vesselsinstead of directly transplanting them into neural tissues.Specifically, donor cells transplanted into a vessel can migrate to theneural tissues and thereby regenerate the neural tissues. Hence, thepresent invention is a breakthrough in developing a therapeutic methodfor relatively noninvasive transplantation.

Furthermore, the present invention adds significantly to elucidate themechanism underlying the differentiation of non-neural cells such ashematopoietic cells and mesenchymal cells into neural cells. When genesdetermining the differentiation are identified and analyzed, use of suchgenes will allow efficient transformation of a sufficient quantity ofnon-neural cells such as hematopoietic cells and mesenchymal cells in aliving body to neural cells. Thus, the present invention is abreakthrough in the field of “gene therapy” for inducing regeneration ofneural tissues.

1. A method for treating neurodegenerative diseases, comprising:injecting an effective amount of cells capable of differentiating intoneural cells or glia cells into a patient afflicted with aneurodegenerative disease, wherein the cells are prepared by a methodcomprising: diluting marrow cells or cord blood cells; isolating amononuclear cell fraction; and selecting cells having surface markersSH2(+), SH3(+), SH4(+), CD29(+), CD44(+), CD14(−), CD34(−), and CD45(−)from the mononuclear cell fraction, and wherein the injected cells treatsaid patient's neurodegenerative disease.